**6.1. Basal levels of CYP1A1 and CYP2B in different species**

54 New Approaches to the Study of Marine Mammals

BX41).

(1mM) and beta-naphthoflavone (20mM), solubilized in acetone (0.1%), at three doses (Table 1), plus an acetone (0.1%) control. Fibroblast cell cultures were processed for the comet assay after Caliani *et al.* [13], with some modifications. The cells were centrifuged at 1000 g for 10 min, then were embedded in agarose (0.5% low-melting agarose) and layered on conventional slides, predipped in 1% normal melting agarose. The slides were immersed into a freshly made lysis solution (2.5 M NaCl, 10 mM Tris, 0.1 M EDTA, 1% Triton X-100, and 10% DMSO, pH 10) for at least 1 h at 4°C in the dark. The slides were then placed on a horizontal electrophoresis tray previously filled with freshly prepared cold alkaline buffer and left for 20 min to allow DNA unwinding. Electrophoresis was performed at 25 V and 300 mA for 20 min. The DNA migration was evaluated at three different pH conditions (pH 13, pH 12.1, pH 8). Slides were then neutralized in Tris (0.4 M, pH 7.5) for 3x5 min and stained with Sybr safe 1:10.000 in TE (10 mM Tris-HCl pH 7.5 and 500 mM EDTA pH 7.5) buffer. A total of 50 cells per slide were examined under epifluorescence at 40X magnification. The amount of DNA damage was evaluated as the percentage of DNA migrating out of the nucleus using an image analyser (Komet 6.0 Software, Kinetic Imaging Ltd.), connected to a fluorescent microscope (Olympus

**6. Interspecies differences in the Mixed Function Oxidase (MFO) as biomarker of exposure of different environmental contaminants** 

CYP2A2, CYP2B2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2) [35].

Fibroblast cell cultures of fin whale (RT2), Bryde's whale (MBE3), sperm whale (PM6), killer whale (MOO12), Risso's dolphin (GGL1), bottlenose dolphin (TurNic), striped dolphin (RT1), long-beaked common dolphin (MDC12) and common dolphin (DDL1) were treated for 48 h with different environmental contaminants and the quantification of the induction of endogenous proteins such as CYP1A1 and CYP2B was used as target of toxicological susceptibility. The presence and the induction of CYP1A1 and CYP2B were evaluated with the indirect immunofluorescence and quantified with the Olympus macro, *DetectIntZ*. CYP1A1 is induced by planar compounds and CYP2B by globular compounds. The treatments were performed with OC mixture; flame retardants; PAHs; and BPA (Table 1). In the total mixture of Arochlor 1260 [25] only the 1.3033% shows a CYP1A1 inductive capacity while the remaining congeners are CYP2B inducers [26, 27, 28, 29, 30]. pp'DDE and pp'DDT are known as CYP2B inducers [31, 32] but an experiment on fibroblast cell culture of sperm whale (PM6) treated only with pp'DDT and pp'DDE has shown a capacity of these compounds to induce also the CYP1A1 (Figure 1). Examining at the bromine substitution patterns in the basic structure of the PBDE molecule, and with the support of the other studies on this topic [33] we can say that in the BDE-MXE mixture, the 18.72% is CYP1A1 inducer and the rest of congeners are CYP2B inducers. Benzo(a)pyrene and betanaphthoflavone are important planar compounds and CYP1A1 inducers [31]. Finally BPA that may be a human-specific inducer of the CYP3A4 gene [34], but many studies have shown that BPA inhibits several P450-dependent monooxygenases activities (CYP1A2,

The first result of these experiments in nine cetacean species was the detection of the presence of CYP1A1 and CYP2B in fibroblast cells of all species, revealed by immunofluorescence (Figure 2A-B); a higher basal expression of both proteins was found in Risso's dolphin (GGL1) and bottlenose dolphin (TurNic), while fin whale (RT2) and sperm whale (PM6) showed the lowest levels of these proteins. The Risso's dolphin (GGL1) and the sperm whale (PM6) have a very similar diet, consisting mostly of squid. Nevertheless, they have a very different basal expression of the two cytochromes. As for the other species, very high levels of CYP1A1 were present in the Bryde's whale (MBE3). This mysticete sampled in the Sea of Cortez showed CYP1A1 levels more than 20 times greater than the other mysticete studied, the Mediterranean fin whale (RT2). Regarding the levels of contaminants detected in the blubber of different species, the bottlenose dolphin (TurNic), stranded along the coasts of the Mediterranean Sea, had very high levels of organochlorine contaminants in its blubber (DDTs = 77.4 μg/g lipid weight (l.w.); PCBs = 262.6 μg/g l.w.) that are potent inducers of CYP2B. In fact, especially in this specimen this cytochrome appears to be markedly higher than the levels shown by other species (Figure 2B). But the sperm whale (PM6) was also a stranded specimen found on the Italian coasts (Mediterranean Sea) having high values of these xenobiotics in the blubber. It seems therefore that this basal activity is more species-specific than related to the geographical location, diet, toxicological status, etc. in which the animals were found.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

*0.1 µg/ml* 

*1 µg/ml* 

*5 µg/ml* 

*25 µg/ml* 

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 57

*0.01 µg/ml* 

**RT2 (fin whale)** 100 125.1 170.3 148.0 104.3 143.6

**MBE3 (Bryde's whale)** 100 31.6 106.2 55.8 / /

**TurNic (bottlenose dolphin)** 100 63.0 96.5 81.4 / /

**MDC12 (long-beaked common dolphin)** 100 325.9 312.1 836.9 / /

*0.05%* 

**RT1 (striped dolphin)** 100 56.4 31.0 49.3 94.1 219.6

**DDL1 (common dolphin)** 100 / / 111.4 117.3 82.9

*0.01 µg/ml* 

**RT2 (fin whale)** 100 112.3 110.1 110.4 116.3 146.5

**MBE3 (Bryde's whale)** N.C. N.C. N.C. N.C. N.C. N.C. **PM6 (sperm whale)** 100 / / 134.6 292.1 212.4 **MOO12 (killer whale)** 100 156.4 300.5 456.0 153.3 104.3 **GGL1 (Risso's dolphin)** 100 / / 70.8 45.2 128.1

**TurNic (bottlenose dolphin)** 100 88.6 136.2 85.7 / /

**MDC12 (long-beaked common dolphin)** 100 205.2 127.6 189.4 / /

**Table 2.** (A-B): Mean values of immunofluorescence of CYP1A1 (A) and CYP2B (B) revealed in cultured fibroblasts of different species treated with OC mixture. The immunofluorescence is expressed as index numbers respect to solvent control. Different colour of box is related to different increase of

**RT1 (striped dolphin)** 100 399.8 756.2 305.7 141.8 368.0

**DDL1 (common dolphin)** 100 / / 112.1 102.0 84.7

*0.1 µg/ml* 

*1 µg/ml* 

*5 µg/ml* 

*25 µg/ml* 

**PM6 (sperm whale)** 100 / / 15.7 31.9 77.4 **MOO12 (killer whale)** 100 427.2 288.6 207.8 696.6 49.0 **GGL1 (Risso's dolphin)** 100 / / 144.5 224.1 104.7

**6.2. CYP1A1 and CYP2B in different species after treatment with OC mixture**

*0.05%* 

numbers, are reported in Table 2A-B.

these proteins. N.C. = no cells.

*A CYP1A1 DMSO* 

*B CYP2B DMSO* 

Results of the mean levels of immunofluorescence of CYP1A1 (A) and CYP2B (B), revealed in cultured fibroblasts of different species treated with OC mixture and expressed as index

**Figure 2.** (A-B): Basal levels of immunofluorescence (AUF/nucleus) of CYP1A1 (A) and CYP2B (B) in fibroblast cells of fin whale (B.p.), Bryde's whale (B.e.), sperm whale (P.m.), killer whale (O.o.), Risso's dolphin (G.g.), bottlenose dolphin (T.t.), striped dolphin (S.c.), long-beaked common dolphin (D.c.) and common dolphin (D.d.).
